Antibody Structure
Antibodies are plasma glycoproteins, which consist of a plurality of polypeptide chains connected by disulphide bridges. A standard antibody consists of two identical heavy immunoglobulin (Ig) chains and two identical light chains. Both antibody chains consist of different protein domains having a length of about 110 amino acid residues, which are composed of 6-sheets in the form of a characteristic immunoglobulin fold. The heavy chain consists of one variable (VH) domain and three or four constant domains (CH1, CH2, CH3, CH4). The light chain consists of one variable (VL) and one constant (CL) domain. The variable portions of the heavy and light chain, in particular the hypervariable complementarity determining regions (CDR), contribute to antigen specificity. The immune system provides a high diversity of antibodies against very different antigens. Antibodies can be assigned to different classes, for example IgM, IgA, IgG, IgE, IgD.
Antibody Fragments and Substructures
Antibody fragments can be obtained by enzymatic cleavage or by recombinant processes. A Fab fragment equipped with the antigen recognition function contains the VH-CH1 domains of the heavy chain and the VL-CL domains of the light chain, connected together via disulphide bridges, while the Fv fragment comprises only the heavy and light chain variable regions. However, these fragments consisting of a plurality of protein subunits can be prepared in biologically active form in an acceptable yield only by complex processes (Read et al. (2007), Appl. Environ. Microbiol. 73: 5088-5096).
A single-chain Fv fragment (scFv) is a small, approximately 28 kDa heavy antigen-binding substructure in the form of a covalent coupling of VH and VL domains via a peptide linker (Hu et al. (1996), Cancer Res 56: 3055-3061). However, the folding efficiency, the sometimes inadequate stability and the toxicity of these structures frequently limit the yield in the preparation of biologically active scFv in bacterial expression systems (Nieba et al. (1997), Protein Eng 10: 435-444).
The term minibody (mini-antibody) refers to an approximately 75 kDa heavy chimeric molecule of an scFv and a hinge region from the heavy chain fused with the CH3 domain which, assisted by the CH3 domain, is assembled to form a bivalent molecule covalently bonded via disulphide bridges of the hinge region (Wu, EP0627932B1). The CH3 domain serves as the dimerisation domain for the production of homodimers (cf. Dubel et al., Biol. Unserer Zeit, Vol. 34, No. 6, p. 372-379, 2004). However, on expression in E. coli, these molecules exhibit low expression rates and proteolytic degradation in the hinge region (Hu et al. (1996), Cancer Research 56: 3055-3061).
Like the minibody, the diabody has two antigen-binding sites. In the diabody, VL and VH domains are connected in the form of a single polypeptide chain to give a divalent and bispecific molecule (Hollinger et al. (1993), PNAS 90: 6444-6448).
Monobodies are chimeric antigen-binding polypeptides, which exhibit hypervariable CDR loops within a fibronectin type III scaffold (Koide, EP0985039B1). These molecules provide a valuable class of novel affinity reagents. However, when CDR loops are transplanted into a heterologous fibronectin scaffold, effector and detection functions of native antibodies are lost.
Nanobodies consist of single-chain antigen-binding VHH domains (variable domains of a heavy chain antibody) and are based on the observation that natural and functional antibodies that consist only of heavy chains are found in camels and llamas (Caserman and Harmers, EP 19930919098). The solubility of human VH domains (heavy chain variable domains) is, however, frequently limited on account of hydrophobic regions, which interact in the intact antibody with regions of the light chain (Barthelemy et al. (2007) J. Biol. Chem. 283: 3639-3654).
Antibodies in Immunodiagnosis
There are numerous immunoassay formats for determining the presence or concentration of a specific antibody, for example against a pathogen, an autoantigen or an allergen, in a biological sample. In general, such assays are directed to the detection of a specific antibody class or a combination of particular antibody classes and use specific internal controls or calibrators. Immunoassays which are suitable, for example, for determining human autoantibodies generally contain a positive control, a negative control and an index calibrator or a gradation of different calibrator concentrations (standard series) for producing a calibration curve, with which antibody concentrations in a sample can be interpolated. Such control and calibration reagents are conventionally prepared by diluting seropositive plasmas or serums in a suitable dilution medium.
For example, the calibrators and controls for the isotype-specific determination of β2-glycoprotein autoantibodies are prepared from the serum of human donors, which contain high concentrations of these autoantibodies of classes IgG, IgM and/or IgA. The use of human seropositive serum or plasma to prepare controls and calibrators is associated with numerous disadvantages, however, such as, for example, the difficulty of acquiring such reagents in large amounts and of suitable quality, differences in the binding characteristics in different batches, heterogeneous polyclonal specificity, heterogeneous isotype composition, presence of pathogens, costs, etc.
Because antibodies bind to antigens with high specificity and affinity, they are of central importance in immunodiagnosis. The size of the natural antibody molecules and their complex structure of a plurality of polypeptide chains with a large number of inter- and intra-domains, interconnections through disulphide bridges as well as glycosylation positions represent a considerable obstacle in the construction and the recombinant expression of specific antibodies.
Hackett et al. (EP1018019 B1) disclose a process for the preparation of reagents for use as calibrators and controls, wherein the reagent is a chimeric monoclonal antibody which comprises heavy and light chain variable regions from a first host species fused to heavy and light chain constant regions from a second host species, which corresponds to that of the antibody to be determined. However, the preparation of these species-chimeric monoclonal antibodies requires a high technical outlay, as is typical of the production of monoclonal antibodies. Although Hackett mentions the theoretical possibility of using as synthetic calibrators also polypeptides which bind specifically to a given ligand and are fused to an antibody region of the desired host species, Hackett does not provide a process by means of which single-chain synthetic polypeptide calibrators can be synthesised.
There is accordingly a continuing need for small, antibody-like molecules which specifically recognise an antigen, form complexes with antibodies against natural immunoglobulins, and can be prepared easily and in a large amount in bacterial expression systems.